Therapeutic uses of BPI protein products in humans with hemorrhage due to trauma

ABSTRACT

Methods and materials for the treatment of humans suffering from hemorrhage due to trauma are provided, in which therapeutically effective amounts of BPI protein products are administered.

This is a continuation-in-part application of U.S. Ser. No. 08/652,292,filed May 23, 1996, now abandoned, incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods and materials fortreating humans suffering from hemorrhage due to trauma, byadministration of bactericidal/permeability-increasing (BPI) proteinproducts.

Acute traumatic hemorrhage, generally requiring immediate surgicalintervention, is a major contributor to morbidity and mortality in theU.S. Bickell et al., New Eng. J. Med., 331:1105-1109 (1994), Tran etal., Surgery, 114:21-30 (1993).! In 1982, there were approximately165,000 deaths in the U.S. due to trauma, with at least two additionalcases of permanent disability for each death. About 50% of thesetraumatic deaths occur immediately, due to direct injury to the centralnervous system, heart, or one of the major blood vessels. Additionalearly deaths, approximately 30%, occur within several hours afterinjury, usually due to uncontrolled hemorrhage. The remaining 20% ofdeaths are so-called "late deaths", occurring during days to weeks afterinjury, due to complications from the traumatic hemorrhage that includeinfection or multiple organ system failure (MOSF) in about 80% of thecases. Trunkey, Sci. Am., 249:28-35 (1983), Trunkey, New Eng. J. Med.,324:1259-1263 (1991).!

Among those patients who survive the immediate resuscitative andsurgical interventions, approximately 10-40% suffer from a variety ofmorbidities, including, for example, systemic inflammation, woundinfections, pneumonia, sepsis, respiratory failure, renal failure,coagulopathy, and pancreatitis. Hemorrhage and transfusion requirementsmay be specifically linked to increased risk of postoperative infection,respiratory complications, and multiorgan system failure Agarwal et al.,Arch. Surg., 128:171-177 (1993), Duke et al., Arch. Surg., 128:1125-1132(1993), Tran et al., supra!.

The causes of these complications from traumatic hemorrhage aremultifactorial and interrelated. Many morbidities may be related tosystemic inflammation following injury. It has also been hypothesizedthat physical trauma to tissue, direct tissue hypoperfusion, andtranslocation of endogenous bacteria and absorption of endotoxin fromthe gut lumen (due to hypoperfusion and/or other injury to thegastrointestinal tract) may play a role in the pathogenesis of thesecomplications. The relevance of these proposed factors in thepathophysiology of the morbidities and late deaths associated with acutehemorrhagic shock in humans, however, is not clear.

Although acute traumatic hemorrhage is one potential cause ofhypovolemic shock (i.e., shock due to decreased intravascular volume),there are numerous other potential causes, such as internal bleeding,e.g., gastrointestinal hemorrhage, intraperitoneal or retroperitonealhemorrhage, hemorrhage into the femoral compartment, intrathoracichemorrhage, aortic dissection and ruptured aortic aneurysm; excessivefluid loss due to, e.g., severe vomiting due to an intestinal or pyloricobstruction, severe diarrhea, sweating, dehydration, excessive urination(due to diabetes mellitus, diabetes insipidus, excessive diuretics, orthe diuretic phase of acute renal failure), peritonitis, pancreatitis,planchnic ischemia, gangrene, burns; vasodilation due to, e.g., nervoussystem damage, anesthesia, ganglionic and adrenergic blockers,barbiturate overdose, poisons; and metabolic, toxic, or humoralvasodilatation, such as acute adrenal insufficiency, or an anaphylacticreaction. Other causes of shock unrelated to circulatory volume lossinclude cardiogenic shock (e.g., acute myocardial infarction, cardiactamponade) and obstructive shock (e.g., acute pulmonary embolism). See,e.g., Manual of Medical Therapeutics, 28th ed., Ewald et al., eds.,Little, Brown and Company, Boston (1995); Cecil's Textbook of Medicine,17th ed., Wyngaarden et al., eds., W.B. Saunders Co., Philadelphia(1985).!

As outlined below in Table I below, a normal individual can rapidly loseup to 20 percent of the blood volume without any signs or symptoms.Limited signs of cardiovascular distress appear with losses up to 30percent of the blood volume, but signs and symptoms of hypovolemic shockgenerally appear when the blood loss exceeds 30 to 40 percent of theblood volume.

                  TABLE I                                                         ______________________________________                                        Percentage                                                                            Amount                                                                of Blood                                                                              Lost                                                                  Volume Lost                                                                           (ml)    Clinical Manifestations                                       ______________________________________                                        10-20%   500-   Usually none, perhaps mild postural hypotension                       1000    and tachycardia in response to exercise;                                      vasovagal syncope may occur in 5% of cases                    20-30%  1000-   Few changes supine; light-headedness and                              1500    hypotension commonly occur when upright;                                      marked tachycardia in response to exertion                    30-40%  1500-   Blood pressure, cardiac output, central venous                        2000    pressure, and urine volume are reduced even                                   when supine; thirst, shortness of breath, clammy                              skin, sweating, clouding of consciousness and                                 rapid, thready pulse may be noted                             40-50%  2000-   Severe shock, often resulting in death                                2500                                                                  ______________________________________                                    

The patient is frequently oliguric, with a urinary output of less than20 mL per hour. Frequently, the physical findings follow a progressivepattern as shock evolves from the early compensated phase to theadvanced stages. In Stage I, physiologic compensatory mechanisms, suchas increased cardiac output or elevated systemic vascular resistance,are effective and minimal clinical symptoms and signs are observed. InStage II, these mechanisms cannot effectively compensate for the bloodvolume loss, and the patient may exhibit hypotension, tachycardia, andhyperventilation. The decreased perfusion of vital organs can result inan altered mental state ranging from agitation to stupor to coma,reduced urinary output, and myocardial ischemia (in patients withcoronary artery disease). The external appearance of the patient alsoreflects excessive sympathetic discharge, with cyanosis, coldness, andclamminess of the skin. In Stage III, which may be irreversible, theexcessive and prolonged reduction of tissue perfusion leads tosignificant alterations in cellular membrane function, aggregation ofblood corpuscles, and "sludging" in the capillaries. Thevasoconstriction which has taken place in the less vital organs in orderto maintain blood pressure in now excessive and has reduced flow to suchan extent that cellular damage occurs.

Following traumatic hemorrhage, conventional therapy is directed atstopping the hemorrhage, combating shock, and restoring the bloodvolume. Prompt fluid resuscitation is preferably given throughlarge-bore catheters placed in large peripheral veins. The pneumaticantishock garment, with sequential inflation of legs and abdominalcompartments to 15-40 mm Hg, may temporally stabilize patients byincreasing peripheral systemic vascular resistance. Restoration of theblood volume may be achieved by intravenous infusion of electrolytesolutions; colloid solutions of plasma protein, albumin, or dextran; orfresh whole blood. In the emergency situation, electrolyte solutions,albumin, or dextran are preferred over fresh whole blood because of thelarge amounts of fluid required, the possible delay in transfusion iftyping and cross-matching are performed, and the possibility of allergictransfusion reactions. When shock is due to hemorrhage, packed red bloodcells should be given as soon as feasible. When hemorrhage is massive,type-specific unmatched blood can be given safely. Rarely, type O bloodmay be needed.

Rapid infusion of Ringer's lactated or normal saline solution is themost widely used fluid therapy following hemorrhage. An initial infusionof two to three times the volume of the estimated blood loss isadministered. Because these solutions are rapidly distributed throughoutthe intravascular and extravascular compartments, they must besupplemented with colloid solutions. When large volumes of electrolytesolutions are infused, patients often develop peripheral edema andelderly patients may develop pulmonary edema.

The colloidal preparations in wide use include a 6 percent solution ofhigh molecular weight dextran (dextran 70), a 10 percent solution of lowmolecular weight dextran (dextran 40), and a 5 percent solution ofalbumin in normal saline. Infusions of dextran 70 produce an initialvolume effect slightly greater than the amount infused. Dextran 70 isslowly cleared over one to two days, allowing time for normalphysiologic mechanisms to replace the volume lost. Dextran 40 has theadvantage of an initial volume effect of nearly twice the amountinfused. The lower molecular weight material is more rapidly cleared,however, and the volume-expanding effect is dissipated by 24 hours,before normal volume replacement mechanisms are maximal. Acute renalfailure has occurred in a few patients receiving dextran 40. With eitherdextran solution, volumes in excess of one liter may interfere withplatelet adhesiveness and the normal coagulation cascade. A solution of5 percent albumin in normal saline has the advantage of producing aknown volume effect in the hypovolemic patient, but this preparation isrelatively costly and time-consuming to prepare. A hypertonic albuminpreparation containing 120 mEq of sodium lactate, 120 mEq of sodiumchloride, and 12.5 grams of albumin per liter provides a predictablevolume effect and minimizes interstitial fluid leakage. Use ofhypertonic solutions requires careful monitoring of arterial and centralvenous pressures to avoid fluid overload. Coexisting problems such ascongestive heart failure, valvular heart disease, myocardial ischemia,or renal insufficiency must be carefully monitored, and invasivehemodynamic monitoring must be considered during acute management.Associated coagulopathy and electrolyte imbalance must also becorrected.

BPI is a protein isolated from the granules of mammalianpolymorphonuclear leukocytes (PMNs or neutrophils), which are bloodcells essential in the defense against invading microorganisms. HumanBPI protein has been isolated from PMNs by acid extraction combined witheither ion exchange chromatography Elsbach, J. Biol. Chem., 254:11000(1979)! or E. coli affinity chromatography Weiss, et al., Blood, 69:652(1987)!. BPI obtained in such a manner is referred to herein as naturalBPI and has been shown to have potent bactericidal activity against abroad spectrum of gram-negative bacteria. The molecular weight of humanBPI is approximately 55,000 daltons (55 kD). The amino acid sequence ofthe entire human BPI protein and the nucleic acid sequence of DNAencoding the protein have been reported in FIG. 1 of Gray et al., J.Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Grayet al. amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat.No. 5,198,541 discloses recombinant genes encoding and methods forexpression of BPI proteins, including BPI holoprotein and fragments ofBPI.

BPI is a strongly cationic protein. The N-terminal half of BPI accountsfor the high net positive charge; the C-terminal half of the moleculehas a net charge of -3. Elsbach and Weiss (1981), supra.! A proteolyticN-terminal fragment of BPI having a molecular weight of about 25 kDpossesses essentially all the anti-bacterial efficacy of thenaturally-derived 55 kD human BPI holoprotein. Ooi et al., J. Bio.Chem., 262: 14891-14894 (1987)!. In contrast to the N-terminal portion,the C-terminal region of the isolated human BPI protein displays onlyslightly detectable anti-bacterial activity against gram-negativeorganisms. Ooi et al., J. Exp. Med., 174:649 (1991).! An N-terminal BPIfragment of approximately 23 kD, referred to as "rBPI₂₃," has beenproduced by recombinant means and also retains anti-bacterial activityagainst gram-negative organisms. Gazzano-Santoro et al., Infect. Immun.60:4754-4761 (1992).

The bactericidal effect of BPI has been reported to be highly specificto gram-negative species, e.g., in Elsbach and Weiss, Inflammation:Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter30, Raven Press, Ltd. (1992). The precise mechanism by which BPI killsgram-negative bacteria is not yet completely elucidated, but it isbelieved that BPI must first bind to the surface of the bacteria throughelectrostatic and hydrophobic interactions between the cationic BPIprotein and negatively charged sites on LPS. In susceptiblegram-negative bacteria, BPI binding is thought to disrupt LPS structure,leading to activation of bacterial enzymes that degrade phospholipidsand peptidoglycans, altering the permeability of the cell's outermembrane, and initiating events that ultimately lead to cell death.Elsbach and Weiss (1992), supra!. LPS has been referred to as"endotoxin" because of the potent inflammatory response that itstimulates, i.e., the release of mediators by host inflammatory cellswhich may ultimately result in irreversible endotoxic shock. BPI bindsto lipid A, reported to be the most toxic and most biologically activecomponent of LPS.

BPI protein has never been used previously for the treatment of humanssuffering from hemorrhage due to trauma or the shock associated withtraumatic blood loss (i.e., hypovolemic shock). Bahrami et al.,presentation at Vienna International Endotoxin Society Meeting, August,1992, report the administration of BPI protein to rats subjected tohemorrhage. Yao et al., Ann. Surg., 221:398-405 (1995), report theadministration of rBPI₂₁ (described infra) to rats subjected toprolonged hemorrhagic insult for 180 minutes followed by resuscitation.U.S. Pat. Nos. 5,171,739, 5,089,724 and 5,234,912 report the use of BPIin various in vitro and in vivo animal model studies asserted to becorrelated to methods of treating endotoxin-related diseases, includingendotoxin-related shock. In co-owned, co-pending U.S. application Ser.Nos. 08/378,228, filed Jan. 24, 1995, 08/291,112, filed Aug. 16, 1994,and 08/188,221, filed Jan. 24, 1994, incorporated herein by reference,the administration of BPI protein product to humans with endotoxin incirculation was described. See also, von der Mohlen et al., J. Infect.Dis. 172:144-151 (1995); von der Mohlen et al., Blood 85:3437-3443(1995); de Winter et al., J. Inflam. 45:193-206 (1995)!. In co-owned,co-pending U.S. application Ser. No. 08/644,287 filed May 10, 1995, theadministration of BPI protein product to humans suffering from severemeningococcemia was described.

In spite of treatment with antibiotics and state-of-the-art medicalintensive care therapy, human mortality and morbidities associated withhemorrhage due to trauma remain significant and unresolved by currenttherapies. New therapeutic methods are needed that could reduce orameliorate the adverse events and improve the clinical outcome of suchpatients.

SUMMARY OF THE INVENTION

The present invention provides novel methods for treating humanssuffering from hemorrhage due to trauma, involving the administration ofBPI protein products to provide clinically verifiable alleviation of theadverse effects of, or complications associated with, this diseasestate, including mortality and complications or morbidities.

According to the invention, BPI protein products such as rBPI₂₁ areadministered to humans suffering from acute traumatic hemorrhage inamounts sufficient to reduce or prevent mortality and/or to reduce theincidence (i.e., occurrence) or severity of complications ormorbidities, including infection (e.g., surgical site infection) ororgan dysfunction (e.g., disseminated intravascular coagulation, acuterespiratory distress syndrome, acute renal failure, or hepatobiliarydysfunction).

Also contemplated is use of a BPI protein product in the preparation ofa medicament for the treatment of humans suffering from hemorrhage dueto trauma.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon consideration of the followingdetailed description of the invention which describes presentlypreferred embodiments thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the incidence of adverse events in rBPI₂₁ and placebotreatment groups.

DETAILED DESCRIPTION

Acute hemorrhage due to trauma is a life-threatening condition withsignificant mortality and morbidities despite state-of-the-art medicalintensive care. The administration of BPI protein products to humanssuffering from hemorrhage due to acute traumatic injury (such aspenetrating and/or blunt trauma) is expected to effectively decreasemortality and reduce the incidence (i.e., occurrence) or severity ofcomplications or morbidities associated with or resulting fromhemorrhage due to trauma. Complications include infection (e.g., insurgical sites, wounds, organs, anatomical spaces, the bloodstream, theurinary tract, or pneumonia) or organ dysfunction (e.g., disseminatedintravascular coagulation, acute respiratory distress syndrome (ARDS),acute renal failure, or hepatobiliary dysfunction), and may includeserious complications. An additional complication may be pulmonarydysfunction, which includes ARDS and pneumonia. These unexpected effectson the mortality and complications associated with and resulting fromhemorrhage due to trauma indicate that BPI protein products effectivelyinterfere with or block a number of the multiple poorly-understoodpathophysiologic processes that have led to poor outcomes in thiscondition. BPI protein products may be used as adjunctive therapy in thetreatment or prevention of organ dysfunction and serious infections. BPIprotein products are expected to provide beneficial effects for patientssuffering from hemorrhage due to trauma, such as reduced injury severityscore, reduced length of time on ventilatory support and inotropic(vasoactive) therapy, reduced duration or severity of associatedcoagulopathy, reduced stay in the ICU, reduced stay in the hospitaloverall, and reduced incidence and duration of complications such ascoagulopathy, respiratory failure, renal failure, hepatic failure, comaor altered mental state, adrenal cortical necrosis, and severeinfection, including in wounds, organs, anatomical spaces, thebloodstream, the urinary tract, or pneumonia.

Therapeutic compositions comprising BPI protein product may beadministered systemically or topically. Systemic routes ofadministration include oral, intravenous, intramuscular or subcutaneousinjection (including into a depot for long-term release), intraocularand retrobulbar, intrathecal, intraperitoneal (e.g. by intraperitonealravage), intrapulmonary using aerosolized or nebulized drug, ortransdermal. The preferred route is intravenous administration. Whengiven parenterally, BPI protein product compositions are generallyinjected in doses ranging from 1 μg/kg to 100 mg/kg per day, preferablyat doses ranging from 0.1 mg/kg to 20 mg/kg per day, more preferably atdoses ranging from 1 to 20 mg/kg/day and most preferably at dosesranging from 2 to 10 mg/kg/day. Treatment may be initiated immediatelyafter the trauma or within a time period subsequent to the trauma(including, e.g., within 6, 12 or 24 hours after trauma, or within aclinically reasonable time period determined by the treating physician,for example, 48 to 72 hours after trauma). Presently preferred is acontinuous intravenous infusion of BPI protein product at a dose of 4 to6 mg/kg/day, continuing for 48 to 72 hours. The treatment may continueby continuous infusion or intermittent injection or infusion, at thesame, reduced or increased dose per day for, e.g., 1 to 3 days, andadditionally as determined by the treating physician. Alternatively, BPIprotein products are administered intravenously by an initial bolusfollowed by a continuous infusion. One such regimen is a 1 to 20 mg/kgintravenous bolus of BPI protein product followed by intravenousinfusion at a dose of 1 to 20 mg/kg/day, continuing for up to one week.Another such dosing regimen is a 2 to 10 mg/kg initial bolus followed byintravenous infusion at a dose of 2 to 10 mg/kg/day, continuing for upto 72 hours. Topical routes include administration in irrigation fluidsfor, e.g., irrigation of wounds, or intrathoracic or intraperitonealcavities. Other topical routes include administration in the form ofsalves, ophthalmic drops, ear drops, or medicated shampoos. For example,for topical administration in drop form, about 10 to 200 μL of a BPIprotein product composition may be applied one or more times per day asdetermined by the treating physician. Those skilled in the art canreadily optimize effective dosages and administration regimens fortherapeutic compositions comprising BPI protein product, as determinedby good medical practice and the clinical condition of the individualpatient.

As used herein, "BPI protein product" includes naturally andrecombinantly produced BPI protein; natural, synthetic, and recombinantbiologically active polypeptide fragments of BPI protein; biologicallyactive polypeptide variants of BPI protein or fragments thereof,including hybrid fusion proteins and dimers; biologically activepolypeptide analogs of BPI protein or fragments or variants thereof,including cysteine-substituted analogs; and BPI-derived peptides. TheBPI protein products administered according to this invention may begenerated and/or isolated by any means known in the art. U.S. Pat. No.5,198,541, the disclosure of which is incorporated herein by reference,discloses recombinant genes encoding and methods for expression of BPIproteins including recombinant BPI holoprotein, referred to as rBPI₅₀(or rBPI) and recombinant fragments of BPI. Co-owned, copending U.S.patent application Ser. No. 07/885,501 and a continuation-in-partthereof, U.S. patent application Ser. No. 08/072,063 filed May 19, 1993and corresponding PCT Application No. 93/04752 filed May 19, 1993, whichare all incorporated herein by reference, disclose novel methods for thepurification of recombinant BPI protein products expressed in andsecreted from genetically transformed mammalian host cells in cultureand discloses how one may produce large quantities of recombinant BPIproducts suitable for incorporation into stable, homogeneouspharmaceutical preparations.

Biologically active fragments of BPI (BPI fragments) includebiologically active molecules that have the same or similar amino acidsequence as a natural human BPI holoprotein, except that the fragmentmolecule lacks amino-terminal amino acids, internal amino acids, and/orcarboxy-terminal amino acids of the holoprotein. Nonlimiting examples ofsuch fragments include a N-terminal fragment of natural human BPI ofapproximately 25 kD, described in Ooi et al., J. Exp. Med., 174:649(1991), and the recombinant expression product of DNA encodingN-terminal amino acids from 1 to about 193 or 199 of natural human BPI,described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992),and referred to as rBPI₂₃. In that publication, an expression vector wasused as a source of DNA encoding a recombinant expression product(rBPI₂₃) having the 31-residue signal sequence and the first 199 aminoacids of the N-terminus of the mature human BPI, as set out in FIG. 1 ofGray et al., supra, except that valine at position 151 is specified byGTG rather than GTC and residue 185 is glutamic acid (specified by GAG)rather than lysine (specified by AAG). Recombinant holoprotein (rBPI₅₀)has also been produced having the sequence (SEQ ID NOS: 1 and 2) set outin FIG. 1 of Gray et al., supra, with the exceptions noted for rBPI₂₃and with the exception that residue 417 is alanine (specified by GCT)rather than valine (specified by GTT). Other examples include dimericforms of BPI fragments, as described in co-owned and co-pending U.S.patent application Ser. No. 08/212,132, filed Mar. 11, 1994, andcorresponding PCT Application No. PCT/US95/03125, the disclosures ofwhich are incorporated herein by reference. Preferred dimeric productsinclude dimeric BPI protein products wherein the monomers areamino-terminal BPI fragments having the N-terminal residues from about 1to 175 to about 1 to 199 of BPI holoprotein. A particularly preferreddimeric product is the dimeric form of the BPI fragment havingN-terminal residues 1 through 193, designated rBPI₄₂ dimer.

Biologically active variants of BPI (BPI variants) include but are notlimited to recombinant hybrid fusion proteins, comprising BPIholoprotein or biologically active fragment thereof and at least aportion of at least one other polypeptide, and dimeric forms of BPIvariants. Examples of such hybrid fusion proteins and dimeric forms aredescribed by Theofan et al. in co-owned, copending U.S. patentapplication Ser. No. 07/885,911, and a continuation-in-part applicationthereof, U.S. patent application Ser. No. 08/064,693 filed May 19, 1993and corresponding PCT Application No. US93/04754 filed May 19, 1993,which are all incorporated herein by reference and include hybrid fusionproteins comprising, at the amino-terminal end, a BPI protein or abiologically active fragment thereof and, at the carboxy-terminal end,at least one constant domain of an immunoglobulin heavy chain or allelicvariant thereof. Similarly configured hybrid fusion proteins involvingpart or all Lipopolysaccharide Binding Protein (LBP) are alsocontemplated for use in the present invention.

Biologically active analogs of BPI (BPI analogs) include but are notlimited to BPI protein products wherein one or more amino acid residueshave been replaced by a different amino acid. For example, co-owned,copending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993and corresponding PCT Application No. US94/01235 filed Feb. 2, 1994, thedisclosures of which are incorporated herein by reference, disclosespolypeptide analogs of BPI and BPI fragments wherein a cysteine residueis replaced by a different amino acid. A preferred BPI protein productdescribed by this application is the expression product of DNA encodingfrom amino acid 1 to approximately 193 or 199 of the N-terminal aminoacids of BPI holoprotein, but wherein the cysteine at residue number 132is substituted with alanine and is designated rBPI₂₁ Δcys or rBPI₂₁.Other examples include dimeric forms of BPI analogs; e.g. co-owned andco-pending U.S. patent application Ser. No. 08/212,132 filed Mar. 11,1994, and corresponding PCT Application No. PCT/US95/03125, thedisclosures of which are incorporated herein by reference.

Other BPI protein products useful according to the methods of theinvention are peptides derived from or based on BPI produced byrecombinant or synthetic means (BPI-derived peptides), such as thosedescribed in co-owned and co-pending U.S. patent application Ser. No.08/504,841 filed Jul. 20, 1995 and in co-owned and copending PCTApplication No. PCT/US94/10427 filed Sep. 15, 1994, which corresponds toU.S. patent application Ser. No. 08/306,473 filed Sep. 15, 1994, and PCTApplication No. US94/02465 filed Mar. 11, 1994, which corresponds toU.S. patent application Ser. No. 08/209,762, filed Mar. 11, 1994, whichis a continuation-in-part of U.S. patent application Ser. No.08/183,222, filed Jan. 14, 1994, which is a continuation-in-part of U.S.patent application Ser. No. 08/093,202 filed Jul. 15, 1993 (for whichthe corresponding international application is PCT Application No.US94/02401 filed Mar. 11, 1994), which is a continuation-in-part of U.S.patent application Ser. No. 08/030,644 filed Mar. 12, 1993, thedisclosures of all of which are incorporated herein by reference.

Presently preferred BPI protein products include recombinantly-producedN-terminal fragments of BPI, especially those having a molecular weightof approximately between 21 to 25 kD such as rBPI₂₃ or rBPI₂₁, ordimeric forms of these N-terminal fragments (e.g., rBPI₄₂ dimer).Additionally, preferred BPI protein products include rBPI₅₀ andBPI-derived peptides. Particularly preferred is rBPI₂₁.

The administration of BPI protein products is preferably accomplishedwith a pharmaceutical composition comprising a BPI protein product and apharmaceutically acceptable diluent, adjuvant, or carrier. The BPIprotein product may be administered without or in conjunction with knownsurfactants, other chemotherapeutic agents or additional knownanti-microbial agents. One pharmaceutical composition containing BPIprotein products (e.g., rBPI₅₀, rBPI₂₃) comprises the BPI proteinproduct at a concentration of 1 mg/ml in citrate buffered saline (5 or20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight ofpoloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc.,Wilmington, Del.). Another pharmaceutical composition containing BPIprotein products (e.g., rBPI₂₁) comprises the BPI protein product at aconcentration of 2 mg/mL in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer188 and 0.002% polysorbate 80. Such combinations are described inco-owned, co-pending PCT Application No. US94/01239 filed Feb. 2, 1994,which corresponds to U.S. patent application Ser. No. 08/190,869 filedFeb. 2, 1994 and U.S. patent application Ser. No. 08/012,360 filed Feb.2, 1993, the disclosures of all of which are incorporated herein byreference.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples. Example 1addresses the effect of BPI protein product administration in humans onthe mortality and complications associated with hemorrhage due totrauma.

EXAMPLE 1 Clinical Study Protocol--Therapeutic Effects of BPI ProteinProduct

A human clinical study was designed to examine the effect of anexemplary BPI protein product, rBPI₂₁, in the treatment of patients withacute hemorrhage due to trauma. Thus, a multicenter, randomized,double-blind, placebo-controlled trial was implemented comparing placebotreatment and rBPI₂₁ treatment given over 48 hours in patients withacute hemorrhage due to trauma. Approximately 400 patients admitted tothe emergency department with acute hemorrhage due to trauma andrequiring transfusion of at least two units of blood were randomized ina 1:1 ratio for treatment with either rBPI₂ or placebo. In addition tostandard therapy, each patient received by continuous intravenousinfusion either rBPI₂₁ at 8 mg/kg over 48 hours (4 mg/kg/day×2 days) orthe equivalent volume of placebo. In most instances the weight of thepatient in kilograms was determined as a best estimate.

Efficacy was monitored from Day 1 to Day 15 by following patients fordevelopment of complications, such as impaired organ function andinfection, and for survival. Safety was monitored by pre-treatment andserial post-treatment testing of chemistries and hematology parameters,as well as daily assessments for adverse events through Day 15. A finalassessment of survival and adverse complications occurred on Day 29.

Patients brought to the hospital with acute hemorrhage due to traumawere selected for enrollment in the study if they met the followinginclusion and exclusion criteria. Inclusion criteria were: (1) age 18(or age of consent) to 75 years, inclusive; (2) patient suffering fromacute hemorrhage secondary to trauma; (3) study drug given within 6 or12 hours of occurrence of the traumatic event (if precise time of eventwas unknown, best estimate was provided); (4) patient requires and hasbegun to receive a second unit of packed red blood cells; and (5)patient provides verbal informed consent or next of kin provides writteninformed consent. Exclusion criteria were: (1) a Triage Revised TraumaScore (IRTS, scale 0-12) less than 2.0 upon admission to the EmergencyDepartment, see Table II below Champion et al., Crit. Care Med.,9(9):672-676 (1981); Greenfield et al., Chapter 10, in SurgeryScientific Principles and Practices, J.B. Lippincott Co., Philadelphia,pp. 252-255 (1993)!; (2) severe head trauma (Glasgow Coma Score (GCS) ≦5or equivalent evidence), see Table III below Teasdale et al., Lancet, 1:81 (1974)!; (3) isolated cranial injury; (4) spinal injury withparalysis; (5) burn injuries with at least 20% body surface area withsecond degree burns; (6) known positive HIV (test not mandatory atentry); (7) known pre-existing renal disease (creatinine >2.0); (8)known pre-existing cardiac disease (NY Heart Association class greaterthan III, see Table IV below Braunwald, in Braunwald et al., HeartDisease, The Textbook of Cardiovascular Medicine, 3rd ed., W.B. SaundersCompany, Philadelphia, Pa., page 12 (1988); J. Am. Med. Ass'n,249:539-544 (1988)!); (9) known pre-existing primary or metastaticmalignancy in visceral organs; (10) arterial pH (at initial evaluation)<6.8 or base deficit >15 (if measured); (11) known current steroidtherapy (>10 mg prednisone/day for>one month); (12) known pre-existingcirrhosis or active hepatitis; (13) pregnancy or lactation; (14)participation in other investigational drug studies (includinginvestigational blood products) within previous 30 days; (15) weight(estimated) greater than 120 kg; and (16) a "do not resuscitate" (DNR)or equivalent order.

                  TABLE II                                                        ______________________________________                                        Triage Revised Trauma Score (TRTS)*                                           ASSESSMENT                                                                              METHOD                                                              ______________________________________                                                                     CODING                                           Respiratory                                                                             Count respiratory rate in 15                                                                     10-29 =  4                                       Rate      sec and multiply by 4                                                                            >29 =    3                                       (RR)                         6-9 =    2                                                                    1-5 =    1                                                                    0 =      0                                       Systolic Blood                                                                          Measure systolic cuff pressure                                                                   >89 =    4                                       Pressure (SBP)                                                                          in either arm by auscultation                                                                    76-89 =  3                                                 or palpation       50-75 =  2                                                                    1-49 =   1                                                                    0 =      0                                       Glasgow Coma                                                                            Calculate according to Table                                                                     Convert GCS to                                   Score     III below          the Following                                    (GCS)                        Code:                                                                   13-15 =                                                                              4                                                                      9-12 = 3                                                                      6-8 =  2                                                                      4-5 =  1                                                                      <4 =   0                                               ______________________________________                                         *The TRTS is the sum of the codes for RR, SBP and GCS (range 0-12).      

                  TABLE III                                                       ______________________________________                                        Glasgow Coma Scale*                                                           ______________________________________                                        Eye Opening                                                                                  Spontaneous    4                                                              Response to sound                                                                            3                                                              Response to pain                                                                             2                                                              Never          1                                               Motor Response                                                                               Obey commands  6                                                              Localized pain 5                                                              Normal flexion 4                                                              (withdrawal)                                                                  Abnormal flexion                                                                             3                                                              (decorticate)                                                                 No response    1                                               Verbal Response                                                                              Oriented       5                                                              Confused conversation                                                                        4                                                              Inappropriate words                                                                          3                                                              Incomprehensible sounds                                                                      2                                                              None           1                                               ______________________________________                                         *Scores range from 3 to 15                                               

                  TABLE IV                                                        ______________________________________                                        Modified New York Heart Association                                           Functional Classification                                                     ______________________________________                                        Class I.                                                                              Patients with cardiac disease but with no limitation of                       physical activity. Ordinary physical activity causes no                       undue dyspnea, anginal pain, fatigue, or palpitation.                 Class IIS.                                                                            Patients with slight limitation of physical activity.                         They are comfortable at rest and with moderate                                exertion. They experience symptoms only with the                              more strenuous grades of ordinary activity.                           Class IIM.                                                                            Patients with moderate limitation of physical ability.                        They are comfortable at rest and with mild exertion.                          They experience symptoms with moderate grades of                              ordinary activity.                                                    Class III.                                                                            Patients with marked limitation of physical activity.                         They are comfortable at rest but experience symptoms                          even with the milder forms of ordinary activity.                      Class IV.                                                                             Patients with inability to carry on any physical activity                     without discomfort. Symptoms of cardiac insufficiency                         or of the anginal syndrome may be present, even at                            rest, and are intensified by activity.                                ______________________________________                                    

The following were recorded for all patients randomized to treatment:(1) date and estimated time of incident, and date and time of admissionto the Emergency Department; (2) for patients randomized and nottreated, the reason for not treating; (3) from arrival at hospital untilapproximately 48 hours postoperatively, date, time, volume, and locationthat the patient received blood, blood products, and fluids such aspacked red blood cells, whole blood, autotransfusion, platelets, freshfrozen plasma, crystalloid, or colloid, at locations such as EmergencyDepartment, Operating Room, Post-anesthesia Care Unit, or SurgicalIntensive Care Unit; however, if the patient did not undergo surgery,the above items that were applicable were collected during study days 1,2, and 3; (4) date and time the second unit of blood was administered(which should have preceded surgery, to assure that hemorrhage is due totrauma, not surgery), and date and start and stop times of anesthesia;(5) date and start and stop times of surgery, estimated blood loss inoperating room, and date and time in post-anesthesia care unit; (6) dateand time study drug infusion began and ended, volume infused, andreasons for temporary or permanent discontinuation; if applicable, andif discontinued, quantity infused; (7) directed medical history(including extent and nature of injuries, intercurrent diseases,conditions contributing to bleeding, etc.), demographic and directedphysical exam information, such as gender, age, weight (estimated ormeasured), height (estimated or measured), vital signs, physical signsof injury; (8) results of the pregnancy test performed during screeningfor eligibility of appropriate female patients (all women of childbearing potential, i.e., all women who were not either surgicallysterile or documented to be post-menopausal); and (9) results of theTRTS performed during screening for eligibility (including actualmeasurements).

After transfusion of the second unit of blood was initiated, theinvestigator administered an unknown test drug from kits in numberedconsecutive order. Each kit contained either rBPI₂₁ or placebo. TherBPI₂₁ was supplied as a clear, colorless, sterile non-pyrogenicsolution in 10 mL single use glass vials at a concentration of 2 mg/mLin 5 mM sodium citrate/0.15M sodium chloride buffer, pH 5.0 with 0.2%poloxamer 188 and 0.002% polysorbate 80, containing no preservative. TherBPI₂₁ was stored refrigerated at 2-8° C. at all times prior toadministration. The placebo was supplied as a clear, colorless sterilenon-pyrogenic solution in 10 mL single use glass vials. It was composedof 0.2 mg/mL human serum albumin in 5 mM sodium citrate/0.15M sodiumchloride buffer, pH 5.0, containing no preservative. The placebo wasalso stored refrigerated at 2-8° C. at all times prior toadministration. The kit assigned to each patient contained a sufficientnumber of vials of study medication for all doses for that patient. Eachvial contained 10 mL of test article.

The study was administered to two groups ("active" rBPI₂₁ and placebocontrol) as outlined above. The study medication was brought to roomtemperature prior to infusion. Throughout the dosing procedure, goodaseptic technique for intravenous administration was followed. The studymedication was administered by intravenous infusion into a central orperipheral vein over 48 hours. The infusion bag/tubing administrationset was completely changed after 24 hours. Suitability of intravenousaccess was determined by easy withdrawal of blood from the access, aswell as easy infusion of intravenous fluids without infiltration. Thestudy medication was the sole agent administered in the chosen portduring the course of the infusion protocol. The venous access port wasnot heparinized, but was flushed as necessary with physiologic saline.Any sign of a reaction at a site of infusion was recorded on thepatient's case record form and source document as an adverse experience.

Patients treated at selected study sites are assessed for: (1) bloodlevels of rBPI₂₁ : blood for the assessment of the rBPI₂₁ level is drawnat the following times (at selected study sites only): prior to thestart of the infusion (up to 60 minutes prior to the start of theinfusion), the following times (hours) after the start of the infusion;1, 4, 8, 12, 20, 24, 32, 36, 40, within 15 minutes prior to thecompletion of the 48 hour infusion, and the following times aftercompletion of the infusion; 7 minutes (48:07), 15 minutes (48:15), 30minutes (48:30), 1 hour (49:00), 3 hours (51:00), 6 hours (54:00), and24 hours (72:00); (2) antibodies to rBPI₂₁ : blood for assessment ofantibodies to rBPI₂₁ is drawn at selected study sites at the followingtimes: Day 1 prior to study drug infusion, and Days 15 and 29, if thepatient is still in hospital or returns to clinic (actual draw days mayvary from Days 10-20 and Days 21-29); and (3) cytokines: blood forassessment of cytolines is drawn at selected study sites.

The following safety laboratory panels were assessed at Day 1 prior totest drug infusion, Day 3 (after end of infusion) and Day 8, however, ifpatient is discharged on or prior to Day 8, assessment was made prior todischarge if possible: (1) hematology panel: hemoglobin, hematocrit,erythrocyte count, leukocyte count and differential, and platelet count;(2) serum chemistry panel: sodium, potassium, chloride, calcium,phosphorous, blood urea nitrogen, creatinine, uric acid, glucose(fasting), CPK, cholesterol, albumin, total protein, AST (SGOT), ALT(SGPT), bilirubin (total), GGT, LDH, and alakaline phosphatase.

The following were recorded for all treated patients through Day 15and/or Day 29 post-initiation of study drug infusion: (1) adverse events(continued through Day 29); (2) survival status including date andcause(s) of death (continued through Day 29); (3) dates in ICU(continued through Day 29); (4) dates in hospital (continued through Day29); (5) dates on ventilator (continued through Day 29); (6) dates ondialysis or hemofiltration, specifying method (continued through Day29); (7) concomitant medications, including daily amounts of bloodtransfused (continued through Day 15 or Day 29); (8) primary surgicalprocedures performed, for example, including re-operations but excludingprocedures like placement of central lines, Swan-Ganz catheters,arterial lines; lumbar punctures, etc. (continued through Day 15); (9)injury severity score (ISS) based on diagnostic evaluations performedduring current hospital stay; (10) daily assessment of organdysfunctions and the presence of infections (continued through Day 15);(11) daily vital signs associated with and including daily maximum anddaily minimum temperatures (continued through Day 15); and (12)inspection of infusion site used for study drug administration at leastevery eight hours, with observations documented in progress notes or theequivalent.

Organ dysfunctions were assessed using the following definitions. Thepatient was considered to have disseminated intravascular coagulation(DIC) when there were: (1) abnormally low values for platelets (or therewas a >25% decrease from a previously documented value) and either anelevated prothrombin time or an elevated partial thromboplastin time andclinical evidence of bleeding, or (2) if obtained, a confirmatory testwas positive (FDP >1:40 or D-Dimers >2.0). These abnormalities must haveoccurred in the absence of medically significant confounding factorssuch as liver failure, major hematoma, or anticoagulant therapy.

The patient was considered to have acute respiratory distress syndrome(ARDS) when: bilateral pulmonary infiltrates consistent with pulmonaryedema were present, and PaO₂ /FiO₂ <200. These signs must have occurredin the absence of congestive heart failure or primary lung disease suchas pulmonary embolus or pneumonia. The Pulmonary Artery Wedge Pressure(PAWP), when measured, must have been <18 mm Hg.

The patient was considered to have acute renal failure (ARF) when: (1)dialysis or hemofiltration was required (definition used for primaryanalysis), or (2) serum creatinine became abnormal with an increaseof >2.0 mg/dL in a patient with documented normal baseline creatinine,or (3) serum creatinine was >3.0 mg/dL in a patient not known to haverenal insufficiency, but whose (pretrauma) baseline creatinine wasunknown, or (4) serum creatinine was doubled from admission orpre-rBPI₂₁ treatment level in a patient with previous renalinsufficiency. These findings must not have been prerenal in nature(e.g. associated with dehydration or gastrointestinal bleeding) or dueto rhabdomyolysis.

Post-surgical hepatobiliary dysfunction (HBD) was evaluated only inpatients without primary hepatic disease (e.g., hepatitis or cirrhosis),alcoholism, or biliary disease. The patient was considered to havehepatobiliary dysfunction when: the bilirubin exceeded 3.0 mg/dL, andeither the alkaline phosphatase, gamma glutamyl transpeptidase (GGT),alanine aminotransferase (ALT, or SGPT) or aspartate aminotransferase(AST, or SGOT) exceeded twice the upper limit of normal. These findingsmust have occurred in the absence of confounding disease.

Patients were also evaluated for infections in wounds, surgical sites(both superficial and deep incisional sites), organs, anatomical spaces,the bloodstream (bacteremia), the urinary tract, or the respiratorytract (pneumonia).

The physician principal investigators were provided with the definitionsof each organ dysfunction and were asked to record at pre-treatment anddaily during Days 1-15 (a) whether each organ dysfunction was "Present","Clinically present", "Not present or clinically present", or "Unknown"according to the definitions provided and (b) all available actuallaboratory or clinical data required by the definition, whether or notthe definition was met. Investigators were also provided with thedefinitions of infections and were asked to record (a) whether each typeof infection was "Present" or "Not present" at any time during Days 1-15and (b) if "Present", the actual culture or clinical data required bythe definition.

In order to provide a more objective analysis of these endpoints, andupon recommendation of an independent Data Safety Monitoring Board,computer programs were developed prior to the first efficacy interimanalysis at 50% accrual to implement the same organ dysfunction andinfection definitions using the actual laboratory, clinical and culturedata required for each definition. The algorithmic approach defined eachorgan dysfunction as "Present," "Clinically present," "Not present orclinically present," or "Unknown" at pretreatment and daily during days1-15. Each organ dysfunction was classified as "Unknown" on any givenday if certain minimum assessments required by the definition were notprovided on that day. The definition for "Present" on a given dayrequired that all assessments had been made and that each assessment metits respective criterion for the specified organ dysfunction. Thus,patients for whom one or more required assessments were missing on agiven day could not be classified as having met the definition for"Present" for that organ dysfunction for that day. On days on whichassessments were incomplete, the organ dysfunction was classified as"Clinically present" if each of the nonmissing assessments met theirrespective criteria for "Present" for that organ dysfunction. Therefore,"Clinically present" implies that the organ dysfunction may have beenpresent that day, based on incomplete evidence, and that nocontradictory evidence was recorded that day. Since serious ARF wasdefined as the use of dialysis/hemofiltration on at least one day duringDays 1-15, "Clinically present" was not applicable to serious ARF. Anorgan dysfunction was considered "Not present or clinically present"when none of the definitions for "Present," "Clinically present" or"Unknown" were met for that day. Patients who were classified as havingan organ dysfunction "Present" or "Clinically present" pretreatment wererequired to have satisfied the primary endpoint by another complicationin order to have been classified as having met the primary endpointduring Days 1-15.

For infections, the algorithmic approach defmed each infection as"Present" or "Not present" during Days 1-15. Each definition for"Present" required that the definition be strictly met on at least oneday during Days 1-15 according to the data provided. If the patient didnot meet the "Present" definition of infection, they were classified as"Not present" for that infection.

Serious complications were defmed as the occurrence of the followingserious infections: (1) a deep incisional surgical site infection, (2)an organ or anatomical space infection, (3) a secondary bloodstreaminfection, (4) a primary bloodstream infection, and (5) pneumonia; orthe following serious organ dysfunctions: (1) disseminated intravascularcoagulation (DIC) or coagulopathy, (2) acute respiratory distresssyndrome (ARDS), (3) acute renal failure (ARF) requiring dialysis orhemofiltration, and (4) hepatobiliary dysfunction (HBD). Patients werecounted once as suffering from complications regardless of the number ofcomplications.

Across 19 sites, 1411 patients were screened and 401 patients wererandomized into the study groups and received therapy. Among these 401patients, 199 received placebo treatment and 202 received rBPI₂₁treatment. Thirty-one patients (15 placebo, 16 rBPI₂₁) did not receivethe complete administration of study drug. Twelve patients (six placeboand six rBPI₂₁) were withdrawn from the study before Day 29. Data fromall patients who received any amount of study medication, even ifinfusion was incomplete, were included in all safety and efficacyanalyses, whether or not the patient was withdrawn from the study.

The mean age of the study population was 35 (range: 16-80 years); 80% ofthe patients were under 45 years of age. The mean dosing weight was 79kg (range: 45-145 kg). Seventy-seven percent of the patients were male.The traumatic injury source was classified as blunt trauma (50%),penetrating trauma (48%), or both (2%). Of the other trauma relatedcharacteristics, the mean TRTS was 10.6 (range: 0-12); the mean GCS was13.2 (range: 3-15); the mean ISS (version '90) was 23.9 (range: 1-75),the mean number of PRBC units started prior to study drug infusion was6.4 (range: 0-57), and the mean time from traumatic incident to druginfusion was 9.5 hours (range: 1.3-21.8 hours). There were no notabletreatment group differences for age, weight, ethnicity, injury source,TRTS, GCS, and the time to infusion (p>0.10 controlling for site), butthe placebo group had a somewhat higher proportion of females (p=0.11,controlling for site). The number of units of packed red blood cells(PRBC) transfused prior to drug infusion was similar between patientsrandomized to rBPI₂₁ and placebo. The mean ISS was slightly worse(p=0.07 controlling for site) for placebo patients (mean 25.1) than forrBP1₂₁ patients (mean 22.7).

The prespecified primary efficacy analysis focused on the primaryendpoint of mortality or serious complication (defined as serious organdysfunction or serious infection as assessed by the algorithmicapproach) occurring at any time after Hour 0 through Day 15. Overallmortality in this study was low (approximately 5-6%). Treatment groupswere compared using Cox regression, stratifying by site and unadjustedfor covariates. The results were analyzed by computer algorithm usingthe strict definitions of "Present" and also after incorporatingincomplete evidence (leading to classifications of "Present orClinically present"). Regardless of the algorithmic method, the rate ofmortality or serious complication by Day 15 was lower by 7% for rBPI₂₁patients compared to placebo patients. The Kaplan-Meier estimates ofevent rates for mortality or serious complication using both algorithmicmethods are shown below in Table V.

                  TABLE V                                                         ______________________________________                                                            Placebo  rBPI.sub.21                                                                            p-                                      Outcomes and Statistical Calculations                                                             (N = 199)                                                                              (N = 202)                                                                              value                                   ______________________________________                                        "Present"                                                                            Actual Event Rate Percentage                                                                   46%      39%                                          Definition                                                                           (#patients with events/                                                                        (91/199) (78/202)                                     by     #total patients)                                                       Algorithm                                                                            Kaplan-Meier Estimates of                                                                      46%      39%    0.17                                         Event Rates at 15 days                                                 "Present/                                                                            Actual Event Rate Percentage                                                                   55%      48%                                          Clinically                                                                           (#patients with events/                                                                        (109/199)                                                                              (97/202)                                     Present"                                                                             #total patients)                                                       Definition                                                                           Kaplan-Meier Estimates of                                                                      55%      48%    0.15                                  by     Event Rates at 15 days                                                 Algorithm                                                                     ______________________________________                                    

Placebo patients were approximately 1.27 times more likely to experiencemortality or serious complication than rBPI₂₁ patients by bothalgorithmic methods as measured by the hazard ratio from Cox regression(rBPI₂₁ to placebo hazard ratio=0.79; p=0.13 for mortality or seriouscomplication using "Present"; p=0.09 for mortality or seriouscomplication using "Present or Clinically Present", stratified bycenter).

As a secondary analysis, the primary analysis was repeated withadjustment for significant covariates, resulting in the following hazardratios: rBPI₂₁ to placebo hazard ratio using "Present"=0.79, p=0.14(adjusting for age, injury source, ISS'90 and units PRBC transfusedprior to drug infusion); hazard ratio using "Present or ClinicallyPresent"=0.81, p=0.16 (adjusting for age, ISS'90 and units PRBCtransfused prior to drug infusion). Event incidence for each of thesecondary efficacy measures assessed is shown in FIG. 1. Analysis ofthese secondary efficacy measures revealed lower frequencies of thefollowing complications in patients treated with rBPI₂₁ compared topatients treated with the placebo preparation: any complication, anyserious complication, any organ dysfunction, any serious organdysfunction, any infection, any serious infection and pneumonia. Slightreductions were also noted in favor of rBPI₂₁ treatment in theproportion of patients developing disseminated intravascular coagulationor coagulopathy, primary and secondary bloodstream infection andasymptomatic bacteriuria.

Adverse events in this severely injured population were frequent inpatients treated with either rBPI₂₁ or placebo. There were, however,numerically higher percentages of patients with adverse events in theplacebo group compared to the rBPI₂₁ group. A higher percentage ofpatients were also noted to experience any extremely abnormalpost-treatment laboratory result in the placebo treated group comparedto the rBPI₂₁ treated group. These data suggest a possible additionalbeneficial effect.

In summary, this controlled clinical trial evaluating a single dosingregimen has demonstrated a trend in favor of rBPI₂₁ treatment in theprimary endpoint of mortality or serious complication through Day 15.Reductions were also noted in the proportion of patients who experiencedcomplications. These results, taken together, are consistent with abeneficial effect for treatment with rBPI₂₁ in patients with hemorrhagedue to trauma.

Numerous modifications and variations of the above-described inventionare expected to occur to those of skill in the art. Accordingly, onlysuch limitations as appear in the appended claims should be placedthereon.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1813 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 31..1491                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 124..1491                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                #"rBPI"   (D) OTHER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - CAGGCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AAC AT - #G GCC AGG GGC          54                                                                          #Glu Asn Met Ala Arg Gly                                                      25                                                                            - CCT TGC AAC GCG CCG AGA TGG GTG TCC CTG AT - #G GTG CTC GTC GCC ATA          102                                                                          Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Me - #t Val Leu Val Ala Ile           10                                                                            - GGC ACC GCC GTG ACA GCG GCC GTC AAC CCT GG - #C GTC GTG GTC AGG ATC          150                                                                          Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gl - #y Val Val Val Arg Ile           #       5  1                                                                  - TCC CAG AAG GGC CTG GAC TAC GCC AGC CAG CA - #G GGG ACG GCC GCT CTG          198                                                                          Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gl - #n Gly Thr Ala Ala Leu           # 25                                                                          - CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GA - #C TAC TCA GAC AGC TTT          246                                                                          Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro As - #p Tyr Ser Asp Ser Phe           #                 40                                                          - AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AG - #C TTC TAC AGC ATG GAC          294                                                                          Lys Ile Lys His Leu Gly Lys Gly His Tyr Se - #r Phe Tyr Ser Met Asp           #             55                                                              - ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG AT - #A AGC ATG GTG CCC AAT          342                                                                          Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Il - #e Ser Met Val Pro Asn           #         70                                                                  - GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AA - #T ATC AAG ATC AGC GGG          390                                                                          Val Gly Leu Lys Phe Ser Ile Ser Asn Ala As - #n Ile Lys Ile Ser Gly           #     85                                                                      - AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA AT - #G AGC GGC AAT TTT GAC          438                                                                          Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Me - #t Ser Gly Asn Phe Asp           #105                                                                          - CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GA - #T CTG AAG CTG GGC AGT          486                                                                          Leu Ser Ile Glu Gly Met Ser Ile Ser Ala As - #p Leu Lys Leu Gly Ser           #               120                                                           - AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC TG - #C TCC AGC TGC AGC AGC          534                                                                          Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cy - #s Ser Ser Cys Ser Ser           #           135                                                               - CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AA - #G AGC AAA GTC GGG TGG          582                                                                          His Ile Asn Ser Val His Val His Ile Ser Ly - #s Ser Lys Val Gly Trp           #       150                                                                   - CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TC - #T GCG CTT CGA AAC AAG          630                                                                          Leu Ile Gln Leu Phe His Lys Lys Ile Glu Se - #r Ala Leu Arg Asn Lys           #   165                                                                       - ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AA - #T TCT GTA TCC TCC AAG          678                                                                          Met Asn Ser Gln Val Cys Glu Lys Val Thr As - #n Ser Val Ser Ser Lys           170                 1 - #75                 1 - #80                 1 -       #85                                                                           - CTG CAA CCT TAT TTC CAG ACT CTG CCA GTA AT - #G ACC AAA ATA GAT TCT          726                                                                          Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Me - #t Thr Lys Ile Asp Ser           #               200                                                           - GTG GCT GGA ATC AAC TAT GGT CTG GTG GCA CC - #T CCA GCA ACC ACG GCT          774                                                                          Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pr - #o Pro Ala Thr Thr Ala           #           215                                                               - GAG ACC CTG GAT GTA CAG ATG AAG GGG GAG TT - #T TAC AGT GAG AAC CAC          822                                                                          Glu Thr Leu Asp Val Gln Met Lys Gly Glu Ph - #e Tyr Ser Glu Asn His           #       230                                                                   - CAC AAT CCA CCT CCC TTT GCT CCA CCA GTG AT - #G GAG TTT CCC GCT GCC          870                                                                          His Asn Pro Pro Pro Phe Ala Pro Pro Val Me - #t Glu Phe Pro Ala Ala           #   245                                                                       - CAT GAC CGC ATG GTA TAC CTG GGC CTC TCA GA - #C TAC TTC TTC AAC ACA          918                                                                          His Asp Arg Met Val Tyr Leu Gly Leu Ser As - #p Tyr Phe Phe Asn Thr           250                 2 - #55                 2 - #60                 2 -       #65                                                                           - GCC GGG CTT GTA TAC CAA GAG GCT GGG GTC TT - #G AAG ATG ACC CTT AGA          966                                                                          Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Le - #u Lys Met Thr Leu Arg           #               280                                                           - GAT GAC ATG ATT CCA AAG GAG TCC AAA TTT CG - #A CTG ACA ACC AAG TTC         1014                                                                          Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Ar - #g Leu Thr Thr Lys Phe           #           295                                                               - TTT GGA ACC TTC CTA CCT GAG GTG GCC AAG AA - #G TTT CCC AAC ATG AAG         1062                                                                          Phe Gly Thr Phe Leu Pro Glu Val Ala Lys Ly - #s Phe Pro Asn Met Lys           #       310                                                                   - ATA CAG ATC CAT GTC TCA GCC TCC ACC CCG CC - #A CAC CTG TCT GTG CAG         1110                                                                          Ile Gln Ile His Val Ser Ala Ser Thr Pro Pr - #o His Leu Ser Val Gln           #   325                                                                       - CCC ACC GGC CTT ACC TTC TAC CCT GCC GTG GA - #T GTC CAG GCC TTT GCC         1158                                                                          Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val As - #p Val Gln Ala Phe Ala           330                 3 - #35                 3 - #40                 3 -       #45                                                                           - GTC CTC CCC AAC TCC TCC CTG GCT TCC CTC TT - #C CTG ATT GGC ATG CAC         1206                                                                          Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Ph - #e Leu Ile Gly Met His           #               360                                                           - ACA ACT GGT TCC ATG GAG GTC AGC GCC GAG TC - #C AAC AGG CTT GTT GGA         1254                                                                          Thr Thr Gly Ser Met Glu Val Ser Ala Glu Se - #r Asn Arg Leu Val Gly           #           375                                                               - GAG CTC AAG CTG GAT AGG CTG CTC CTG GAA CT - #G AAG CAC TCA AAT ATT         1302                                                                          Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Le - #u Lys His Ser Asn Ile           #       390                                                                   - GGC CCC TTC CCG GTT GAA TTG CTG CAG GAT AT - #C ATG AAC TAC ATT GTA         1350                                                                          Gly Pro Phe Pro Val Glu Leu Leu Gln Asp Il - #e Met Asn Tyr Ile Val           #   405                                                                       - CCC ATT CTT GTG CTG CCC AGG GTT AAC GAG AA - #A CTA CAG AAA GGC TTC         1398                                                                          Pro Ile Leu Val Leu Pro Arg Val Asn Glu Ly - #s Leu Gln Lys Gly Phe           410                 4 - #15                 4 - #20                 4 -       #25                                                                           - CCT CTC CCG ACG CCG GCC AGA GTC CAG CTC TA - #C AAC GTA GTG CTT CAG         1446                                                                          Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Ty - #r Asn Val Val Leu Gln           #               440                                                           - CCT CAC CAG AAC TTC CTG CTG TTC GGT GCA GA - #C GTT GTC TAT AAA             1491                                                                          Pro His Gln Asn Phe Leu Leu Phe Gly Ala As - #p Val Val Tyr Lys               #           455                                                               - TGAAGGCACC AGGGGTGCCG GGGGCTGTCA GCCGCACCTG TTCCTGATGG GC - #TGTGGGGC       1551                                                                          - ACCGGCTGCC TTTCCCCAGG GAATCCTCTC CAGATCTTAA CCAAGAGCCC CT - #TGCAAACT       1611                                                                          - TCTTCGACTC AGATTCAGAA ATGATCTAAA CACGAGGAAA CATTATTCAT TG - #GAAAAGTG       1671                                                                          - CATGGTGTGT ATTTTAGGGA TTATGAGCTT CTTTCAAGGG CTAAGGCTGC AG - #AGATATTT       1731                                                                          - CCTCCAGGAA TCGTGTTTCA ATTGTAACCA AGAAATTTCC ATTTGTGCTT CA - #TGAAAAAA       1791                                                                          #               1813ATG TG                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 487 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Arg Glu Asn Met Ala Arg Gly Pro Cys As - #n Ala Pro Arg Trp Val         20                                                                            - Ser Leu Met Val Leu Val Ala Ile Gly Thr Al - #a Val Thr Ala Ala Val         # 1                                                                           - Asn Pro Gly Val Val Val Arg Ile Ser Gln Ly - #s Gly Leu Asp Tyr Ala         #              15                                                             - Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Gl - #u Leu Lys Arg Ile Lys         #         30                                                                  - Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Ly - #s His Leu Gly Lys Gly         #     45                                                                      - His Tyr Ser Phe Tyr Ser Met Asp Ile Arg Gl - #u Phe Gln Leu Pro Ser         # 65                                                                          - Ser Gln Ile Ser Met Val Pro Asn Val Gly Le - #u Lys Phe Ser Ile Ser         #                 80                                                          - Asn Ala Asn Ile Lys Ile Ser Gly Lys Trp Ly - #s Ala Gln Lys Arg Phe         #             95                                                              - Leu Lys Met Ser Gly Asn Phe Asp Leu Ser Il - #e Glu Gly Met Ser Ile         #       110                                                                   - Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Th - #r Ser Gly Lys Pro Thr         #   125                                                                       - Ile Thr Cys Ser Ser Cys Ser Ser His Ile As - #n Ser Val His Val His         130                 1 - #35                 1 - #40                 1 -       #45                                                                           - Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gl - #n Leu Phe His Lys Lys         #               160                                                           - Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Se - #r Gln Val Cys Glu Lys         #           175                                                               - Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pr - #o Tyr Phe Gln Thr Leu         #       190                                                                   - Pro Val Met Thr Lys Ile Asp Ser Val Ala Gl - #y Ile Asn Tyr Gly Leu         #   205                                                                       - Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Le - #u Asp Val Gln Met Lys         210                 2 - #15                 2 - #20                 2 -       #25                                                                           - Gly Glu Phe Tyr Ser Glu Asn His His Asn Pr - #o Pro Pro Phe Ala Pro         #               240                                                           - Pro Val Met Glu Phe Pro Ala Ala His Asp Ar - #g Met Val Tyr Leu Gly         #           255                                                               - Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Le - #u Val Tyr Gln Glu Ala         #       270                                                                   - Gly Val Leu Lys Met Thr Leu Arg Asp Asp Me - #t Ile Pro Lys Glu Ser         #   285                                                                       - Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Th - #r Phe Leu Pro Glu Val         290                 2 - #95                 3 - #00                 3 -       #05                                                                           - Ala Lys Lys Phe Pro Asn Met Lys Ile Gln Il - #e His Val Ser Ala Ser         #               320                                                           - Thr Pro Pro His Leu Ser Val Gln Pro Thr Gl - #y Leu Thr Phe Tyr Pro         #           335                                                               - Ala Val Asp Val Gln Ala Phe Ala Val Leu Pr - #o Asn Ser Ser Leu Ala         #       350                                                                   - Ser Leu Phe Leu Ile Gly Met His Thr Thr Gl - #y Ser Met Glu Val Ser         #   365                                                                       - Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Ly - #s Leu Asp Arg Leu Leu         370                 3 - #75                 3 - #80                 3 -       #85                                                                           - Leu Glu Leu Lys His Ser Asn Ile Gly Pro Ph - #e Pro Val Glu Leu Leu         #               400                                                           - Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Le - #u Val Leu Pro Arg Val         #           415                                                               - Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pr - #o Thr Pro Ala Arg Val         #       430                                                                   - Gln Leu Tyr Asn Val Val Leu Gln Pro His Gl - #n Asn Phe Leu Leu Phe         #   445                                                                       - Gly Ala Asp Val Val Tyr Lys                                                 450                 4 - #55                                                   __________________________________________________________________________

What is claimed is:
 1. A method of treating a pulmonary dysfunctioncomplication in a human suffering from hemorrhage due to traumacomprising the step of administering a therapeutically effective amountof a bactericidal/permeability-increasing (BPI) protein product to saidhuman.
 2. The method of claim 1 wherein the BPI protein product is anamino-terminal fragment of BPI protein having a molecular weight ofabout 21 kD to 25 kD.
 3. The method of claim 1 wherein the BPI proteinproduct is rBPI₂₃ or a dimeric form thereof.
 4. The method of claim 1wherein the BPI protein product is rBPI₂₁.
 5. The method of claim 1wherein said pulmonary dysfunction complication is pneumonia.
 6. Themethod of claim 1 wherein the human suffering from hemorrhage due totrauma is additionally administered at least two units of packed redblood cells.
 7. The method of claim 1 wherein the BPI protein product isadministered to said human before development of the pulmonarydysfunction complication.